丹酚酸B对NASH细胞焦亡通路NLRP3/Caspase-1/GSDMD的影响

Effect of salvianolic acid B on NLRP3/caspase-1/GSDMD pyrolysis pathway in NASH cells

背景大量实验证明丹酚酸B(salvianolic acid B,Sal B)对非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)有治疗作用,细胞焦亡在NAFLD发病及其向非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)进展的过程中起了重要作用,通过研究Sal B对NASH细胞焦亡主要相关蛋白的调节,发觉Sal B在NAFLD中多途径、多靶点的治疗作用及优势.目的建立NASH体外细胞模型,并用Sal B及焦亡抑制剂(VX-765)干预,探讨Sal B对细胞焦亡的影响及对焦亡通路GSDMD/NLRP3/Caspase-1的调节机制.方法取对数生长期HepG2细胞,MTT法检测筛选棕榈酸(palmitic acid,PA)最佳的药物干预浓度及干预时间;将细胞随机分为对照组、模型组(PA),治疗组(Sal B+PA)及抑制剂组(VX-765+PA).取各组细胞分别经油红O染色在光学显微镜观察细胞内脂质蓄积情况,收集细胞上清液酶标仪下测定光密度(optical density,OD)值;免疫荧光H2DCFH探针测定细胞内活性氧含量;酶联免疫吸附试验(enzyme-linked immunosorbent assays,ELISA)检测各组细胞上清液白细胞介素-18(interleukin 18,IL-18)、白细胞介素1β(interleukin 1β,IL-1β)表达量;Western Blot检测炎性小体核苷酸寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)及焦亡相关蛋白半胱氨酸蛋白酶-1(Caspase-1)、消皮素(gasdermin D,GSDMD)、GSDMD的N端结构域(GSDMD-N)的表达,并检测各种蛋白的相对表达量变化.结果模型组细胞内脂质小体及活性氧(reactive oxygen species,ROS)蓄积明显,IL-1β、IL-18等促炎因子分泌增多(P值<0.05),炎症小体NLRP3、焦亡相关蛋白Caspase-1、GSDMD、GSDMD-N表达均升高(P值均<0.05).使用Sal B干预治疗或VX-765抑制剂处理后可逆转PA造成的脂质及ROS蓄积,IL-1β及IL-18炎症因子的分泌,同时可下调NLRP3、Caspase-1、GSDMD、GSDMD-N等焦亡相关蛋白表达(P值均<0.05).结论PA可诱导肝脏细胞焦亡发生,丹酚酸B通过对焦亡通路NLRP3/Caspase-1/GSDMD的抑制作用减轻炎症反应,缓解NASH进展.

BACKGROUND Numerous studies have proved that salvianolic acid B(Sal B)has a therapeutic effect on non-alcoholic fatty liver disease(NAFLD).Cell pyrolysis plays an important role in the onset of NAFLD and its progression to NASH.By studying the regulatory effect of Sal B on the main proteins related to cell pyrolysis in NASH,it was found that Sal B has the advantages of multiple approaches and multiple targets in the treatment of NAFLD.AIM To establish a non-alcoholic steatohepatitis(NASH)cell model by treating HepG2 cells with palmitic acid(PA),and explore the effect of Sal B on cell pyrolysis and the pyrolysis-related GSDMD/NLRP3/caspase-1 pathway.METHODS HepG2 cells in logarithmic growth phase were collected,and the optimal drug intervention concentration and intervention time for PA were assessed by MTT assay.The cells were randomly divided into a control group,a model group(PA),a treatment group(Sal B+PA),and a pyrolysis inhibitor group(VX-765+PA).The cells of each group were stained with oil red O to observe the lipid accumulation in the cells under an inverted phase-contrast microscope.The cell supernatant was collected to measure the OD value with a microplate reader.The immunofluorescence probe DCFH-DA was used to determine the intracellular ROS content.The expression of interleukin 18(IL-18)and interleukin 1β(IL-1β)in cell supernatant was determined by ELISA.The expression of NLRP3 and pyrolysis-related proteins caspase-1,GSDMD,and GSDMD-N was detected by Western blot.RESULTS Compared with the control group,the model group had obvious accumulation of lipids and ROS,increased secretion of inflammatory factors IL-1βand IL-18(P<0.05),and increased protein expression of NLRP3,caspase-1,GSDMD,and GSDMD-N(P<0.05).Sal B or VX-765 inhibitor treatment can reverse the accumulation of lipids and ROS caused by PA,decreased the secretion of IL-1βand IL-18,and down-regulated the protein expression of NLRP3,caspase-1,GSDMD,and GSDMD-N(P<0.05).CONCLUSION PA can induce pyrolysis of liver cells.Sal B can reduce inflammation and alleviate the progression of NASH by inhibiting the NLRP3/caspase-1/GSDMD pathway.